12  Gene Enrichment Analysis III

Removed Ribosomal &Mitochondria-Related Genes from DESeq2 Results

Published

November 7, 2025

To identify biological pathways and processes affected by Cre-mediated ApoE4→ApoE2 conversion in liver tissue, we performed complementary enrichment analyses using the high-confidence differentially expressed genes from Method 1 (combined batches, no batch adjustment).

Removed 16 mitochondrial genes and 146 ribosomal genes from the dataset.

12.0.1 Gene Set Enrichment Analysis (GSEA)

pathway NES padj size
GOBP_MITOCHONDRIAL_RESPIRATORY_CHAIN_COMPLEX_ASSEMBLY 2.056 0.018 96

Gene Set Enrichment Analysis (GSEA) of the complete ranked gene list, excluding mitochondrial and ribosomal genes, identified enrichment of the mitochondrial respiratory chain complex assembly in the liver upon ApoE2 switch (NES = 2.056, padj = 0.018).

There are 50 leading-edge genes driving the enrichment of the GOBP_MITOCHONDRIAL_RESPIRATORY_CHAIN_COMPLEX_ASSEMBLY, including Ndufb4b, Pet117, Ndufb4c, Ndufb1, Ndufb3, Ndufb8, Ndufaf2, Lyrm2, Ndufs4, Pet100, among others._All these genes are nuclear-encoded mitochondrial proteins involved in electron transport and ATP synthesis_ and are upregulated in liver following ApoE4→ApoE2 conversion.

12.1 Brain

12.1.1 Gene Set Enrichment Analysis (GSEA)

pathway NES padj size
GOBP_EPOXYGENASE_P450_PATHWAY 2.236 0.030 26
GOBP_REGULATION_OF_CHROMATIN_ORGANIZATION 2.218 0.013 35
GOBP_ARACHIDONIC_ACID_METABOLIC_PROCESS 2.210 0.011 59
GOBP_XENOBIOTIC_METABOLIC_PROCESS 2.139 0.006 98
GOBP_SPLICEOSOMAL_COMPLEX_ASSEMBLY 2.121 0.029 49
GOBP_POSITIVE_REGULATION_OF_MRNA_CATABOLIC_PROCESS 1.889 0.038 91
GOBP_CELLULAR_RESPONSE_TO_XENOBIOTIC_STIMULUS 1.860 0.029 153
GOBP_POSITIVE_REGULATION_OF_MRNA_METABOLIC_PROCESS 1.855 0.045 125
GOBP_DENDRITE_MORPHOGENESIS 1.854 0.011 188
GOBP_ACTION_POTENTIAL 1.815 0.013 160
GOBP_REGULATION_OF_MRNA_METABOLIC_PROCESS 1.813 0.006 274
GOBP_RESPONSE_TO_ALCOHOL 1.778 0.045 164
GOBP_DENDRITE_DEVELOPMENT 1.711 0.010 315
GOBP_SYNAPSE_ASSEMBLY 1.609 0.045 267
GOBP_CHROMATIN_REMODELING 1.519 0.010 472
GOBP_MRNA_PROCESSING 1.460 0.044 453
GOBP_OXIDATIVE_PHOSPHORYLATION -2.060 0.045 124

GSEA of brain tissue revealed 17 significantly enriched pathways.

12.1.1.1 Reducing Redundant Pathways with collapsePathways

Because many pathways shared the same leading-edge genes, we used the collapsePathways function from the fgsea package to remove redundancy and isolate distinct biological themes. This method groups pathways with overlapping gene sets and keeps only the most significantly enriched representative from each cluster, ensuring that the analysis reflects independent biological processes rather than repetitive signals.

pathway NES padj size
GOBP_REGULATION_OF_CHROMATIN_ORGANIZATION 2.218 0.013 35
GOBP_ARACHIDONIC_ACID_METABOLIC_PROCESS 2.210 0.011 59
GOBP_SPLICEOSOMAL_COMPLEX_ASSEMBLY 2.121 0.029 49
GOBP_CELLULAR_RESPONSE_TO_XENOBIOTIC_STIMULUS 1.860 0.029 153
GOBP_ACTION_POTENTIAL 1.815 0.013 160
GOBP_REGULATION_OF_MRNA_METABOLIC_PROCESS 1.813 0.006 274
GOBP_RESPONSE_TO_ALCOHOL 1.778 0.045 164
GOBP_DENDRITE_DEVELOPMENT 1.711 0.010 315
GOBP_SYNAPSE_ASSEMBLY 1.609 0.045 267
GOBP_CHROMATIN_REMODELING 1.519 0.010 472
GOBP_OXIDATIVE_PHOSPHORYLATION -2.060 0.045 124

This analysis identified 11 representative pathways that summarize the main biological processes affected by hepatic ApoE2 expression. The refined pathway set highlighted significant changes in chromatin organization, lipid metabolism, synaptic remodeling, and mitochondrial function in brain tissue.

Notably, genes related to oxidative phosphorylation showed reduced expression in the brain, in contrast to their increase in the liver, indicating a distinct metabolic adjustment driven by altered ApoE signaling.

There are 74 leading-edge genes driving the enrichment of the GOBP_MITOCHONDRIAL_RESPIRATORY_CHAIN_COMPLEX_ASSEMBLY, including Ndufs7, Cox7a1, Atp5f1e, Cox6a1, Cox4i2, Cox8a, Ndufa3, Ndufa7, Ndufa11, Cox7a2, among others._All these genes encode mitochondrial proteins involved in electron transport and ATP synthesis_ and are downregulated in the brain following ApoE4→ApoE2 conversion.

12.2 Common Leading-Edge Genes in Liver and Brain

Leading-edge analysis revealed several mitochondrial genes as main contributors to pathway enrichment in both liver and brain. To pinpoint shared molecular mechanisms underlying the effects of ApoE isoform conversion, we compared the overlapping leading-edge genes between the two tissues.

This analysis identified 12 mitochondrial genes commonly altered in both liver and brain following ApoE4→ApoE2 conversion: Uqcc2, Ndufb4, Ndufb9, Ndufb2, Ndufs5, Ndufs8, Ndufb6, Ndufs6, Ndufb8, Ndufb7, Sdhaf2, Ndufb5

tissue gene log2FoldChange padj
brain Ndufb2 -0.433 0.000
liver Ndufb2 0.294 0.055
brain Ndufb4 -0.503 0.000
liver Ndufb4 0.561 0.001
brain Ndufb5 -0.211 0.000
liver Ndufb5 0.259 0.002
brain Ndufb6 -0.344 0.000
liver Ndufb6 0.384 0.000
brain Ndufb7 -0.256 0.000
liver Ndufb7 0.341 0.004
brain Ndufb8 -0.270 0.000
liver Ndufb8 0.765 0.000
brain Ndufb9 -0.465 0.000
liver Ndufb9 0.379 0.016
brain Ndufs5 -0.417 0.000
liver Ndufs5 0.267 0.055
brain Ndufs6 -0.341 0.000
liver Ndufs6 0.346 0.005
brain Ndufs8 -0.384 0.000
liver Ndufs8 0.280 0.004
brain Sdhaf2 -0.212 0.000
liver Sdhaf2 0.254 0.015
brain Uqcc2 -0.545 0.000
liver Uqcc2 0.386 0.030

These genes showed opposite patterns of regulation, increasing in the liver while decreasing in the brain. This contrast indicates that hepatic ApoE2 expression influences mitochondrial function in a tissue-specific manner, likely shaping the broader metabolic adjustments observed in this model.

12.3 Analysis of Brain metabolic Genes

To understand how hepatic ApoE2 expression influences brain energy metabolism, we analyzed genes involved in glucose transport, glycolysis, lactate shuttling, glycogen turnover, and mitochondrial function. Most genes in these pathways showed modest but consistent shifts, revealing a coordinated metabolic adjustment rather than isolated changes.

log2FoldChange indicates differential expression in brain following hepatic ApoE4→ApoE2 conversion (i.e., ApoE2 effect).

12.3.1 Glycolysis Core Genes

Several glycolytic enzymes, including Aldoa, Pgk1, Eno1, Ldha, and Aldoc, showed reduced expression, suggesting a slower glycolytic flux. In contrast, Hk1 increased, indicating stronger glucose trapping within cells. Together, these changes suggest a transition from rapid anaerobic glycolysis toward more regulated and efficient glucose utilization.

gene log2FoldChange padj
Ldhb -0.272 0.000
Aldoa -0.354 0.000
Pgk1 -0.209 0.000
Eno1 -0.261 0.000
Aldoc -0.237 0.002
Ldha -0.299 0.009
Pgam1 -0.124 0.010
Hk1 0.213 0.016
Eno3 -0.479 0.017
Gapdh -0.122 0.021
Pkm -0.108 0.082
Gck -0.427 0.110
Pfkp 0.114 0.172
Pklr -0.733 0.385
Hk3 -0.281 0.421
Pfkl -0.103 0.456
Pgam2 0.120 0.607
Aldob 0.237 0.673
Hk2 0.028 0.890
Eno2 0.008 0.912
Pfkm -0.002 0.980

12.3.2 Glucose Transporter Genes

Among glucose transporter genes, Slc2a1, Slc2a6, and Slc2a8 showed reduced expression in brain tissue following hepatic ApoE4→ApoE2 conversion, while Slc2a3 displayed a mild increase. Other transporters, including Slc2a4, Slc5a1, and Slc2a2, did not show significant changes.

gene log2FoldChange padj
Slc2a6 -0.465 0.000
Slc2a1 -0.288 0.002
Slc2a8 -0.298 0.004
Slc2a3 0.174 0.080
Slc2a4 -0.182 0.355
Slc5a1 -0.241 0.431
Slc2a2 0.296 NA

12.3.3 Pentose Phosphate Pathway Genes

Within the pentose phosphate pathway, Tkt showed decreased expression in brain tissue following hepatic ApoE4→ApoE2 conversion. The remaining genes analyzed, Rpia, Pgd, and Rpe, did not display significant expression changes.

gene log2FoldChange padj
Tkt -0.225 0.000
Rpia 0.136 0.214
Pgd -0.013 0.879
Rpe 0.001 0.996

12.3.4 Pyruvate metabolism Genes

Lower Pdk2 and higher Pdp1 and Pdp2 expression reopen the pyruvate dehydrogenase complex, enabling glucose-derived pyruvate to enter the TCA cycle and support oxidative metabolism.

gene log2FoldChange padj
Pdp1 0.207 0.001
Pdk2 -0.193 0.001
Pdk3 0.184 0.001
Pck2 -0.318 0.002
Dld 0.115 0.007
Dlat 0.176 0.022
Pdp2 0.233 0.022
Pdhx 0.096 0.040
Pdha1 0.114 0.094
Pdk1 0.114 0.237
Pdk4 0.107 0.369
Pdhb 0.038 0.393
Pck1 1.001 NA

12.3.5 Lactate Transporter Genes

Lactate transporter genes reflected a rebalancing of the astrocyte–neuron metabolic coupling. The reduction of Slc16a3 (MCT4) and the increase of Slc16a7 (MCT2) suggest less lactate export from astrocytes and greater neuronal uptake, restoring a healthy lactate shuttle.

gene log2FoldChange padj
Slc16a7 0.436 0.002
Slc16a14 0.454 0.004
Slc16a3 -0.431 0.015
Slc16a8 -0.606 0.023
Slc16a11 -0.429 0.049
Slc16a10 0.234 0.101
Slc16a1 -0.012 0.936
Slc16a13 0.015 0.938

12.3.6 Glycogen Metabolism Genes

Regulators of glycogen metabolism, such as Pygm, Agl, and Phka2, also adjusted in line with this more stable metabolic state, showing decreased mobilization of glycogen stores.

gene log2FoldChange padj
Pygm -0.235 0.010
Agl 0.164 0.035
Phka2 0.209 0.039
Gbe1 0.125 0.151
Phkg1 -0.109 0.383
Pygl -0.107 0.423
Pygb -0.034 0.650
Gys1 -0.043 0.653
Phka1 -0.022 0.828
Aga -0.008 0.956
Gys2 -0.149 NA

12.3.7 Metabolic Regulators

12.3.8 11.3.7 Metabolic Regulators

Several metabolic regulators changed expression in brain tissue following hepatic ApoE4→ApoE2 conversion. Insr, Gsk3b, Prkaa2, Pik3ca, Akt3, Irs1, Pik3cb, and Prkaa1 were upregulated, while Akt1, Prkag1, Akt2, and Rheb were downregulated. Plin2 also decreased in expression. Other genes, including Mtor, Irs2, Prkab1, Stk11, Prkag2, Prkab2, and Gsk3a, showed minor or nonsignificant changes.

gene log2FoldChange padj
Insr 0.358 0.000
Gsk3b 0.453 0.000
Akt1 -0.215 0.000
Prkag1 -0.253 0.000
Akt2 -0.237 0.000
Prkaa2 0.348 0.001
Pik3ca 0.262 0.001
Akt3 0.289 0.002
Irs1 0.286 0.004
Pik3cb 0.216 0.007
Prkaa1 0.196 0.019
Plin2 -0.231 0.023
Depdc5 0.139 0.031
Rheb -0.160 0.034
Mtor 0.148 0.061
Irs2 0.153 0.074
Prkab1 -0.154 0.093
Stk11 -0.050 0.352
Prkag2 0.068 0.500
Prkab2 0.053 0.689
Gsk3a -0.024 0.690

12.3.9 Mitochondrial Transport Genes

Mitochondrial transporter genes (Mpc1, Mpc2, Slc25a1, Slc25a10) declined, indicating lower demand for emergency substrate import.

gene log2FoldChange padj
Slc25a10 -0.495 0.000
Slc25a1 -0.435 0.000
Slc25a11 -0.383 0.000
Mpc2 -0.276 0.000
Mpc1 -0.206 0.001

12.3.10 All metabolic pathways (only significant genes )

This section lists all genes with significant expression changes across major metabolic pathways in brain tissue after hepatic ApoE4→ApoE2 conversion.

id gene log2FoldChange padj
glucose_transporters Slc2a6 -0.465 0.000
glucose_transporters Slc2a1 -0.288 0.002
glucose_transporters Slc2a8 -0.298 0.004
glyco_core Ldhb -0.272 0.000
glyco_core Aldoa -0.354 0.000
glyco_core Pgk1 -0.209 0.000
glyco_core Eno1 -0.261 0.000
glyco_core Aldoc -0.237 0.002
glyco_core Ldha -0.299 0.009
glyco_core Pgam1 -0.124 0.010
glyco_core Hk1 0.213 0.016
glyco_core Eno3 -0.479 0.017
glyco_core Gapdh -0.122 0.021
glycogen_metabolism Pygm -0.235 0.010
glycogen_metabolism Agl 0.164 0.035
glycogen_metabolism Phka2 0.209 0.039
lactate_transport Slc16a7 0.436 0.002
lactate_transport Slc16a14 0.454 0.004
lactate_transport Slc16a3 -0.431 0.015
lactate_transport Slc16a8 -0.606 0.023
lactate_transport Slc16a11 -0.429 0.049
metabolic_regulators Insr 0.358 0.000
metabolic_regulators Gsk3b 0.453 0.000
metabolic_regulators Akt1 -0.215 0.000
metabolic_regulators Prkag1 -0.253 0.000
metabolic_regulators Akt2 -0.237 0.000
metabolic_regulators Prkaa2 0.348 0.001
metabolic_regulators Pik3ca 0.262 0.001
metabolic_regulators Akt3 0.289 0.002
metabolic_regulators Irs1 0.286 0.004
metabolic_regulators Pik3cb 0.216 0.007
metabolic_regulators Prkaa1 0.196 0.019
metabolic_regulators Plin2 -0.231 0.023
metabolic_regulators Depdc5 0.139 0.031
metabolic_regulators Rheb -0.160 0.034
mitochondrial_transport Slc25a10 -0.495 0.000
mitochondrial_transport Slc25a1 -0.435 0.000
mitochondrial_transport Slc25a11 -0.383 0.000
mitochondrial_transport Mpc2 -0.276 0.000
mitochondrial_transport Mpc1 -0.206 0.001
pentose_phosphate_pathway Tkt -0.225 0.000
pyruvate_metabolism Pdp1 0.207 0.001
pyruvate_metabolism Pdk2 -0.193 0.001
pyruvate_metabolism Pdk3 0.184 0.001
pyruvate_metabolism Pck2 -0.318 0.002
pyruvate_metabolism Dld 0.115 0.007
pyruvate_metabolism Dlat 0.176 0.022
pyruvate_metabolism Pdp2 0.233 0.022
pyruvate_metabolism Pdhx 0.096 0.040

The data show that the ApoE4 mouse brain lives under chronic metabolic stress, a condition reversed by hepatic ApoE2 expression. The ApoE4 state is marked by glucose hypometabolism, which forces astrocytes to accumulate small lipid droplets stabilized by high Plin2 expression. This is not a resting phase but an inefficient, oxygen-hungry state where fatty acids are oxidized incompletely, producing metabolic intermediates (Farmer, Kluemper, and Johnson 2019). An accompanying rise in Slc2a1, Slc2a6, Slc2a8, Mpc1, Mpc2, Slc25a1, and Slc25a10 may reflect a failed attempt to compensate for the energy shortage. Hepatic ApoE2 expression restores balance, normalizing these transport systems.

This correction involves a deeper reprogramming of glucose metabolism. In ApoE4 brains, glycolysis runs fast but inefficiently, shunting pyruvate to lactate through Ldha and the astrocytic exporter Slc16a3 (MCT4). With hepatic ApoE2, Hk1 rises, trapping glucose in cells, while glycolytic enzymes such as Aldoa, Pgk1, and Eno1 fall. At the same time, Pdk2 declines and Pdp1 and Pdp2 increase, reopening the pyruvate dehydrogenase complex and steering metabolism toward aerobic respiration. This shift calms glycolytic activity and reduces glycogen breakdown, shown by lower Pygm expression.

The lactate shuttle also resets. Astrocytic lactate export drops with reduced Ldha and Slc16a3, while neuronal uptake improves through Slc16a7 (MCT2). The fall in Tkt suggests that oxidative stress decreases as the pentose phosphate pathway relaxes.

Finally, key regulators of energy balance respond. Insr and Irs1 rise, restoring insulin sensitivity, while Plin2 declines, signaling relief from lipid stress. The downregulation of oxidative phosphorylation genes confirms that the brain’s metabolism has cooled from a high-output, inefficient state to a steady, aerobic equilibrium. In sum, hepatic ApoE2 expression resolves the metabolic strain of ApoE4, returning the brain to an efficient and balanced homeostasis.